Compounds

ABSTRACT

The invention provides histidine kinase polypeptides and DNA (RNA) encoding histidine kinase polypetides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing histidine kinase polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

This application is a divisional U.S. application Ser. No. 09/046,086,filed Mar. 23, 1998 now U.S. Pat. No. 6,127,147.

This application claims benefit to U.S. Provisional Patent ApplicationNo. 60/043,489, filed Apr. 10, 1997.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, in these and inother regards, the invention relates to novel polynucleotides andpolypeptides of the histidine kinase family, hereinafter referred to as“histidine kinase”.

BACKGROUND OF THE INVENTION

It is particularly preferred to employ Staphylococcal genes and geneproducts as targets for the development of antibiotics. TheStaphylococci make up a medically important genera of microbes. They areknown to produce two types of disease, invasive and toxigenic. Invasiveinfections are characterized generally by abscess formation effectingboth skin surfaces and deep tissues. S. aureus is the second leadingcause of bacteremia in cancer patients. Osteomyelitis, septic arthritis,septic thrombophlebitis and acute bacterial endocarditis are alsorelatively common. There are at least three clinical conditionsresulting from the toxigenic properties of Staphylococci. Themanifestation of these diseases result from the actions of exotoxins asopposed to tissue invasion and bacteremia. These conditions include:Staphylococcal food poisoning, scalded skin syndrome and toxic shocksyndrome.

The frequency of Staphylococcus aureus infections has risen dramaticallyin the past 20 years. This has been attributed to the emergence ofmultiply antibiotic resistant strains and an increasing population ofpeople with weakened immune systems. It is no longer uncommon to isolateStaphylococcus aureus strains which are resistant to some or all of thestandard antibiotics. This has created a demand for both newanti-microbial agents and diagnostic tests for this organism.

Histidine kinases are components of the TCSTS which autophosphorylate ahistidine residue. The phosphate group is then transferred to thecognate reponse regulator. The Histidine kinases have five shortconserved amino acid sequences (Stock, J. B., Ninfa, A. J.& Stock, A.M.(1989) Microbiol. Rev. 53, 450-490, Swanson, R. V., Alex, L. A. &Simon, M. I.(1994) TIBS 19 485-491). These are the histidine residue,which is phosphorylated, followed after approximately 100 residues by aconserved asparagine residue. After another 15 to 45 residues a DXGXGmotif is found, followed by a FXXF motif after another 10-20 residues.10-20 residues further on another glycine motif, GXG is found. The twoglycine motifs are thought to be involved in nucleotide binding. Thisfamily of histidine kinases includes UhpB protein from Salmonellatyphimurium, which is part of the TCSTS which regulates the uptake ofhexose phosphates (Island, M. D., Wei, B. Y. & Kadner, R. J. (1992) J.Bacteriol. 174, 2754-2762). Response regulators are components of theTCSTS. These proteins are phosphorylated by histidine kinases and inturn once phosphorylated effect the response, often through a DNAbinding domain becoming activated. The response regulators arecharacterized by a conserved N-terminal domain of approximately 100amino acids. The N-terminal domains of response regulators as well asretaining five functionally important residues, corresponding to theresidues D12, D13, D57, T87, K109 in CheY (Matsumura, P., Rydel, J. J.,Linzmeier, R. & Vacante, D. (1984) J. Bacteriol. 160, 36-41), haveconserved structural features (Volz, K. (1993) Biochemistry 32,11741-11753). The 3-dimensional structures of CheY from Salmonellatyphimurium (Stock, A. M., Mottonen, J. M., Stock, J. B.& Schutt, C. E.(1989) Nature, 337, 745-749) and Escherichia coli (Volz, K. & Matsumura,P. (1991) J. Biol. Chem. 266, 15511-15519) and the N-terminal domain ofnitrogen regulatory protein C from S.typhimurium (Volkman, B. F.,Nohaile, M. J., Amy, N. K., Kustu, S. & Wemmer, D. E. (1995)Biochemistry, 34 1413-1424), are available, as well as the secondarystructure of SpoOF from Bacillus subtilis (Feher, V. A., Zapf, J. W.,Hoch, J. A., Dahlquist, F. W., Whiteley, J. M. & Cavanagh, J. (1995)Protein Science, 4, 1801-1814). These structures have a (a/b)5 fold.Several structural residues are conserved between different responseregulator sequences, specifically hydrophobic residues within theβ-sheet hydrophobic core and sites from the a-helices.

Among the processes regulated by TCSTS are production of virulencefactors, motility, antibiotic resistance and cell replication.Inhibitors of TCSTS proteins would prevent the bacterium fromestablishing and maintaining infection of the host by preventing it fromproducing the necessary factors for pathogenesis and thereby haveutility in anti-bacterial therapy.

Clearly, there is a need for factors, such as the novel compounds of theinvention, having a present benefit of being useful to screen compoundsfor antibiotic activity. Such factors may also be used to determinetheir role in pathogenesis of infection, dysfunction and disease. Thereis also a need for identification and characterization of such factorsand their antagonists and agonists which can play a role in preventing,ameliorating or correcting infections, dysfunctions or diseases.

The polypeptides of the invention have amino acid sequence homology to aknown UhpB protein from Salmonella typhimurium.

Each patent application to which this invention claims priority ishereby incorporated by reference in its entirety.

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have beenidentified as novel histidine kinase polypeptides by homology betweenthe amino acid sequence set out in FIG. 2 [SEQ ID NO: 2] and a knownamino acid sequence or sequences of other proteins such as UhpB proteinfrom S. typhimurium.

It is a further object of the invention to provide polynucleotides thatencode histidine kinase polypeptides, particularly polynucleotides thatencode the polypeptide herein designated histidine kinase.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding histidine kinase polypeptidescomprising the sequence set out in FIG. 1 [SEQ ID NO:1], or a variantthereof. In particular, the invention provides the open reading frame(nucleotides 38-1126, inclusive) and the sequences that encode thecorresponding histidine kinase expressed by Staphylococcal bacteria.

In another particularly preferred embodiment of the invention there is anovel histidine kinase protein from Staphylococcus aureus comprising theamino acid sequence of FIG. 2 [SEQ ID NO:2], or a variant thereof.

In accordance with another aspect of the invention there is provided anisolated nucleic acid molecule encoding a mature polypeptide expressibleby the Staphylococcus aureus WCUH 29 strain contained in NCIMB DepositNo. 40771.

A further aspect of the invention there are provided isolated nucleicacid molecules encoding histidine kinase, particularly Staphylococcusaureus histidine kinase, including mRNAs, cDNAs, genomic DNAs. Furtherembodiments of the invention include biologically, diagnostically,prophylactically, clinically or therapeutically useful variants thereof,and compositions comprising the same.

In accordance with another aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of histidine kinase and polypeptides encodedthereby.

Another aspect of the invention there are provided novel polypeptides ofStaphylococcus aureus referred to herein as histidine kinase as well asbiologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

Among the particularly preferred embodiments of the invention arevariants of histidine kinase polypeptide encoded by naturally occurringalleles of the histidine kinase gene.

In a preferred embodiment of the invention there are provided methodsfor producing the aforementioned histidine kinase polypeptides.

In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of the invention, thereare provided products, compositions and methods for (i) assessinghistidine kinase expression, (ii) treating disease, for example,disease, such as, infections of the upper respiratory tract (e.g.,otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis),lower respiratory (e.g., empyema, lung abscess), cardiac (e.g.,infective endocarditis), gastrointestinal (e.g., secretory diarrhoea,splenic absces, retroperitoneal abscess), CNS (e.g., cerebral abscess),eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis,preseptal and orbital cellulitis, darcryocystitis), kidney and urinarytract (e.g., epididymitis, intrarenal and perinephric absces, toxicshock syndrome), skin (e.g., impetigo, folliculitis, cutaneousabscesses, cellulitis, wound infection, bacterial myositis) bone andjoint (e.g., septic arthritis, osteomyelitis), (iii) assaying geneticvariation, (iv) and administering a histidine kinase polypeptide orpolynucleotide to an organism to raise an immunological response againsta bacteria, especially a Staphylococcus aureus bacteria.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided polynucleotides thathybridize to histidine kinase polynucleotide sequences, particularlyunder stringent conditions.

In certain preferred embodiments of the invention there are providedantibodies against histidine kinase polypeptides.

In other embodiments of the invention there are provided methods foridentifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypetideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

In accordance with yet another aspect of the invention, there areprovided histidine kinase agonists and antagonists, preferablybacteriostatic or bacteriocidal agonists and antagonists.

In a further aspect of the invention there are provided compositionscomprising a histidine kinase polynucleotide or a histidine kinasepolypeptide for administration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings depict certain embodiments of the invention. Theyare illustrative only and do not limit the invention otherwise disclosedherein.

FIG. 1 shows the polynucleotide sequence of Staphylococcus aureushistidine kinase [SEQ ID NO:1].

FIG. 2 shows the amino acid sequence of Staphylococcus aureus histidinekinase [SEQ ID NO:2] deduced from the polynucleotide sequence of FIG. 1.

FIG. 3 shows the polynucleotide sequence of Staphylococcus aureus UhpBresponse regulator [SEQ ID NO:5].

FIG. 4 shows the amino acid sequence of Staphylococcus aureus UhpBresponse regulator [SEQ ID NO:6] deduced from the polynucleotidesequence of FIG. 3.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

“Host cell” is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, as thecase may be, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GCG program package (Devereux, J.,et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, andFASTA (Atschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990). TheBLAST X program is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). The well knownSmith Waterman algorithm may also be used to determine identity.

Parameters for polypeptide sequence comparison include the following:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

Parameters for polynucleotide comparison include the following:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

Available as: The “gap” program from Genetics Computer Group, MadisonWis. These are the default parameters for nucleic acid comparisons.

A preferred meaning for “identity” for polynucleotides and polypeptides,as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the referencesequence of SEQ ID NOS:1 or 3, wherein said polynucleotide sequence maybe identical to the reference sequence of SEQ ID NOS: 1 or 3 or mayinclude up to a certain integer number of nucleotide alterations ascompared to the reference sequence, wherein said alterations areselected from the group consisting of at least one nucleotide deletion,substitution, including transition and transversion, or insertion, andwherein said alterations may occur at the 5′ or 3′ terminal positions ofthe reference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among the nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence, and wherein said number of nucleotide alterations isdetermined by multiplying the total number of nucleotides in SEQ IDNOS:1 or 3 by the integer defining the percent identity divided by 100and then subtracting that product from said total number of nucleotidesin SEQ ID NOS:1 or 3, or:

n _(n) ≦x _(n)−(x _(n) ·y),

wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NOS:1 or 3, y is 0.50 for 50%,0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%,0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n). Alterations of a polynucleotide sequence encoding thepolypeptide of SEQ ID NOS:2 or 4 may create nonsense, missense orframeshift mutations in this coding sequence and thereby alter thepolypeptide encoded by the polynucleotide following such alterations.

By way of example, a polynucleotide sequence of the present inventionmay be identical to the reference sequence of SEQ ID NOS:2 or 4, that isit may be 100% identical, or it may include up to a certain integernumber of amino acid alterations as compared to the reference sequencesuch that the percent identity is less than 100% identity. Suchalterations are selected from the group consisting of at least onenucleic acid deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference polynucleotide sequenceor anywhere between those terminal positions, interspersed eitherindividually among the nucleic acids in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofnucleic acid alterations for a given percent identity is determined bymultiplying the total number of amino acids in SEQ ID NOS:2 or 4 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNOS:2 or 4, or:

n _(n) ≦x _(n)−(x _(n) ·y),

wherein n_(n) is the number of amino acid alterations, x_(n) is thetotal number of amino acids in SEQ ID NOS:2 or 4, y is, for instance0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., • is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n).

(2) Polypeptide embodiments further include an isolated polypeptidecomprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97or 100% identity to a polypeptide reference sequence of SEQ ID NOS:2 or4, wherein said polypeptide sequence may be identical to the referencesequence of SEQ ID NO: 2 or may include up to a certain integer numberof amino acid alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least oneamino acid deletion, substitution, including conservative andnon-conservative substitution, or insertion, and wherein saidalterations may occur at the amino- or carboxy-terminal positions of thereference polypeptide sequence or anywhere between those terminalpositions, interspersed either individually among the amino acids in thereference sequence or in one or more contiguous groups within thereference sequence, and wherein said number of amino acid alterations isdetermined by multiplying the total number of amino acids in SEQ IDNOS:2 or 4 by the integer defining the percent identity divided by 100and then subtracting that product from said total number of amino acidsin SEQ ID NOS:2 or 4, or:

n _(a) ≦x _(a)−(x _(a) ·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NOS:2 or 4, y is 0.50 for 50%,0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%,0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

By way of example, a polypeptide sequence of the present invention maybe identical to the reference sequence of SEQ ID NOS:2 or 4, that is itmay be 100% identical, or it may include up to a certain integer numberof amino acid alterations as compared to the reference sequence suchthat the percent identity is less than 100% identity. Such alterationsare selected from the group consisting of at least one amino aciddeletion, substitution, including conservative and non-conservativesubstitution, or insertion, and wherein said alterations may occur atthe amino- or carboxy-terminal positions of the reference polypeptidesequence or anywhere between those terminal positions, interspersedeither individually among the amino acids in the reference sequence orin one or more contiguous groups within the reference sequence. Thenumber of amino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in SEQ ID NOS:2 or 4 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNOS:2 or 4, or:

n _(a) ≦x _(a)−(x _(a) ·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NOS:2 or 4, y is, for instance0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and • is the symbol forthe multiplication operator, and wherein any non-integer product ofx_(a) and y is rounded down to the nearest integer prior to subtractingit from x_(a).

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein.

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

“Polypeptide(s)” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. “Polypeptide(s)” refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. “Polypeptide(s)” includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992) each of which is incorporated by reference herein in itsentirety. Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

“Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

DESCRIPTION OF THE INVENTION

The invention relates to novel histidine kinase polypeptides andpolynucleotides as described in greater detail below. In particular, theinvention relates to polypeptides and polynucleotides of a novelhistidine kinase gene of Staphylococcus aureus, which is related byamino acid sequence homology to UhpB polypeptide from S.typhimurium. Theinvention relates especially to histidine kinase having the nucleotideand amino acid sequences set out in FIG. 1 [SEQ ID NO: 1] and FIG. 2[SEQ ID NO: 2] respectively, and to the histidine kinase nucleotidesequences of the DNA deposited in NCIMB Deposit No. 40771 and amino acidsequences encoded thereby.

Deposited materials

A deposit containing a Staphylococcus aureus WCUH 29 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (NCIMB), 23 St. Machar Drive, Aberdeen AB2 1RY, Scotlandon Sep. 11, 1995 and assigned NCIMB Deposit No. 40771. TheStaphylococcus aureus strain deposit is referred to herein as “thedeposited strain” or as “the DNA of the deposited strain.”

The deposited material is a strain that contains the full lengthhistidine kinase DNA, referred to as “NCIMB 40771” upon deposit. Thesequence of the polynucleotides contained in the deposited material, aswell as the amino acid sequence of the polypeptide encoded thereby, arecontrolling in the event of any conflict with any description ofsequences herein.

The deposit has been made under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Micro-organisms for Purposesof Patent Procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. The deposit is provided merely as convenience to those of skillin the art and is not an admission that a deposit is required forenablement, such as that required under 35 U.S.C. §112.

A license may be required to make, use or sell the deposited materials,and no such license is hereby granted.

Polypeptides

The polypeptides of the invention include the polypeptide of FIG. 2 [SEQID NO:2] (in particular the mature polypeptide) as well as polypeptidesand fragments, particularly those which have the biological activity ofhistidine kinase, and also those which have at least 70% identity to thepolypeptide of FIG. 2 [SEQ ID NO:2] or the relevant portion, preferablyat least 80% identity to the polypeptide of FIG. 2 [SEQ ID NO:2], andmore preferably at least 90% similarity (more preferably at least 90%identity) to the polypeptide of FIG. 2 [SEQ ID NO:2] and still morepreferably at least 95% similarity (still more preferably at least 95%identity) to the polypeptide of FIG. 2 [SEQ ID NO:2] and also includeportions of such polypeptides with such portion of the polypeptidegenerally containing at least 30 amino acids and more preferably atleast 50 amino acids.

A polypeptide having an amino acid sequence having at least, forexample, 95% identity to a reference amino acid sequence of SEQ ID NO:2is intended to mean that an amino acid sequence of the polypeptide isidentical to the reference sequence except that the polypeptide sequencemay include up to five amino acid alterations per each 100 amino acidsof the reference amino acid of SEQ ID NO: 2. In other words, to obtain apolypeptide having an amino acid sequence at least 95% identical to areference amino acid sequence, up to 5% of the amino acid residues inthe reference sequence may be deleted or substituted with another aminoacid, or a number of amino acids up to 5% of the total amino acidresidues in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at theamino or carboxy terminal positions of the reference amino acid sequenceor anywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence.

A fragment is a variant polypeptide having an amino acid sequence thatentirely is the same as part but not all of the amino acid sequence ofthe aforementioned polypeptides. As with histidine kinase polypeptidesfragments may be “free-standing,” or comprised within a largerpolypeptide of which they form a part or region, most preferably as asingle continuous region, a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of the amino acid sequence of FIG. 2 [SEQ ID NO:2], or ofvariants thereof, such as a continuous series of residues that includesthe amino terminus, or a continuous series of residues that includes thecarboxyl terminus. Degradation forms of the polypeptides of theinvention in a host cell, particularly a Staphylococcus aureus, are alsopreferred. Further preferred are fragments characterized by structuralor functional attributes such as fragments that comprise alpha-helix andalpha-helix forming regions, beta-sheet and beta-sheet-forming regions,turn and turn-forming regions, coil and coil-forming regions,hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions.

Also preferred are biologically active fragments which are thosefragments that mediate activities of histidine kinase, including thosewith a similar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Staphylococcusaureus or the ability to initiate, or maintain cause disease in anindividual, particularly a human.

Variants that are fragments of the polypeptides of the invention may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of theinvention.

The histidine kinase of the invention is cognated to the responseregulator having the amino acid sequence disclosed in FIG. 4 [SEQ IDNO:6] and that is encoded by the polynucleotide of FIG. 3 [SEQ ID NO:5].The amino acid sequence of SEQ ID NO 6 is the translated open readingframe of SEQ ID NO:5 which displays homology (28% identity) to the DegUresponse regulator from Bacillus subtilis. The invention provides thatthis response regulator is the response regulator cognate of thehistidine kinase of the invention. This response regulator is useful inmany of the compound screening assays of the invention.

Polynucleotides

Another aspect of the invention relates to isolated polynucleotides thatencode the histidine kinase polypeptide having the deduced amino acidsequence of FIG. 2 [SEQ ID NO:2] and polynucleotides closely relatedthereto and variants thereof.

Using the information provided herein, such as the polynucleotidesequence set out in FIG. 1 [SEQ ID NO:1], a polynucleotide of theinvention encoding histidine kinase polypeptide may be obtained usingstandard cloning and screening methods, such as those for cloning andsequencing chromosomal DNA fragments from bacteria using Staphylococcusaureus WCUH 29 cells as starting material, followed by obtaining a fulllength clone. For example, to obtain a polynucleotide sequence of theinvention, such as the sequence given in FIG. 1 [SEQ ID NO:1], typicallya library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29in E. coli or some other suitable host is probed with a radiolabeledoligonucleotide, preferably a 17-mer or longer, derived from a partialsequence. Clones carrying DNA identical to that of the probe can then bedistinguished using stringent conditions. By sequencing the individualclones thus identified with sequencing primers designed from theoriginal sequence it is then possible to extend the sequence in bothdirections to determine the full gene sequence. Conveniently, suchsequencing is performed using denatured double stranded DNA preparedfrom a plasmid clone. Suitable techniques are described by Maniatis, T.,Fritsch, E. F. and Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989), which is incorporated by reference herein in itsentirety. (see in particular Screening By Hybridization 1.90 andSequencing Denatured Double-Stranded DNA Templates 13.70). Illustrativeof the invention, the polynucleotide set out in FIG. 1 [SEQ ID NO:1] wasdiscovered in a DNA library derived from Staphylococcus aureus WCUH 29.

The DNA sequence set out in FIG. 1 [ SEQ ID NO:1] contains an openreading frame encoding a protein having about the number of amino acidresidues set forth in FIG. 2 [SEQ ID NO:2] with a deduced molecularweight that can be calculated using amino acid residue molecular weightvalues well known in the art. Histidine kinase of the invention isstructurally related to other proteins of the histidine kinase family,as shown by the results of sequencing the DNA encoding histidine kinaseof the deposited strain. The protein exhibits greatest homology to UhpBprotein from S.typhimurium among known proteins. Histidine kinase ofFIG. 2 [SEQ ID NO:2] has about 23% identity over its entire length andabout 55% similarity over its entire length with the amino acid sequenceof UhpB polypeptide from S.typhimurium.

The invention provides a polynucleotide sequence identical over itsentire length to the coding sequence in FIG. 1 [SEQ ID NO:1]. Alsoprovided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro- protein sequence. Thepolynucleotide may also contain non-coding sequences, including forexample, but not limited to non-coding 5′ and 3′ sequences, such as thetranscribed, non-translated sequences, termination signals, ribosomebinding sites, sequences that stabilize mRNA, introns, polyadenylationsignals, and additional coding sequence which encode additional aminoacids. For example, a marker sequence that facilitates purification ofthe fused polypeptide can be encoded. In certain embodiments of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc.Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA tag (Wilson et al.,Cell 37: 767 (1984), incorporated by reference herein in its entirety.Polynucleotides of the invention also include, but are not limited to,polynucleotides comprising a structural gene and its naturallyassociated sequences that control gene expression.

The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Staphylococcus aureus histidinekinase having the amino acid sequence set out in FIG. 2 [SEQ ID NO:2].The term also encompasses polynucleotides that include a singlecontinuous region or discontinuous regions encoding the polypeptide (forexample, interrupted by integrated phage or an insertion sequence orediting) together with additional regions, that also may contain codingand/or non-coding sequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide having thededuced amino acid sequence of FIG. 2 [SEQ ID NO:2]. Variants that arefragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodinghistidine kinase variants, that have the amino acid sequence ofhistidine kinase polypeptide of FIG. 2 [SEQ ID NO:2] in which several, afew, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues aresubstituted, deleted or added, in any combination. Especially preferredamong these are silent substitutions, additions and deletions, that donot alter the properties and activities of histidine kinase.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding histidine kinase polypeptide having the amino acid sequence setout in FIG. 2 [SEQ ID NO:2], and polynucleotides that are complementaryto such polynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identicalover its entire length to a polynucleotide encoding histidine kinasepolypeptide of the deposited strain and polynucleotides complementarythereto. In this regard, polynucleotides at least 90% identical overtheir entire length to the same are particularly preferred, and amongthese particularly preferred polynucleotides, those with at least 95%are especially preferred. Furthermore, those with at least 97% arehighly preferred among those with at least 95%, and among these thosewith at least 98% and at least 99% are particularly highly preferred,with at least 99% being the more preferred.

As an illustration, by a polynucleotide having a nucleotide sequencehaving at least, for example, 95% “identity” to a reference nucleotidesequence of SEQ ID NO: 1 is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1. Inother words, to obtain a polynucleotide having a nucleotide sequence atleast 95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These mutations of the reference sequence may occur at the 5′or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among nucleotides in the reference sequence or in one ormore contiguous groups within the reference sequence.

Preferred embodiments are polynucleotides that encode polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by the DNA of FIG. 1 [SEQ ID NO:1].

The invention further relates to polynucleotides that hybridize to theherein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms “stringent conditions” and “stringent hybridizationconditions” mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the hybridization support in0.1×SSC at about 65° C. Hybridization and wash conditions are well knownand exemplified in Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularlyChapter 11 therein, the disclosure of which is hereby incorporated inits entirety by reference.

The invention also provides a polynucleotide consisting essentially of apolynucleotide sequence obtainable by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO:1 under stringent hybridization conditions with a probe havingthe sequence of said polynucleotide sequence set forth in SEQ ID NO:1 ora fragment thereof; and isolating said DNA sequence. Fragments usefulfor obtaining such a polynucleotide include, for example, probes andprimers described elsewhere herein.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding histidinekinase and to isolate cDNA and genomic clones of other genes that have ahigh sequence similarity to the histidine kinase gene. Such probesgenerally will comprise at least 15 bases. Preferably, such probes willhave at least 30 bases and may have at least 50 bases. Particularlypreferred probes will have at least 30 bases and will have 50 bases orless.

For example, the coding region of the histidine kinase gene may beisolated by screening using the DNA sequence provided in SEQ ID NO: 1 tosynthesize an oligonucleotide probe. A labeled oligonucleotide having asequence complementary to that of a gene of the invention is then usedto screen a library of cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for disease, particularly human disease,as further discussed herein relating to polynucleotide assays.

Polynucleotides of the invention that are oligonucleotides derived fromthe sequences of SEQ ID NOS:1 and/or 2 may be used in the processesherein as described, but preferably for PCR, to determine whether or notthe polynucleotides identified herein in whole or in part aretranscribed in bacteria in infected tissue. It is recognized that suchsequences will also have utility in diagnosis of the stage of infectionand type of infection the pathogen has attained.

The invention also provides.polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, host cells, expression

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof or polynucleotides ofthe invention. Introduction of a polynucleotide into the host cell canbe effected by methods described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), suchas, calcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, enterococci E. coli, streptomycesand Bacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic Assays

This invention is also related to the use of the histidine kinasepolynucleotides of the invention for use as diagnostic reagents.Detection of histidine kinase in a eukaryote, particularly a mammal, andespecially a human, will provide a diagnostic method for diagnosis of adisease. Eukaryotes (herein also “individual(s)”), particularly mammals,and especially humans, infected with an organism comprising thehistidine kinase gene may be detected at the nucleic acid level by avariety of techniques.

Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA or cDNA may also be used in the same ways. Usingamplification, characterization of the species and strain of prokaryotepresent in an individual, may be made by an analysis of the genotype ofthe prokaryote gene. Deletions and insertions can be detected by achange in size of the amplified product in comparison to the genotype ofa reference sequence. Point mutations can be identified by hybridizingamplified DNA to labeled histidine kinase polynucleotide sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase digestion or by differences in melting temperatures.DNA sequence differences may also be detected by alterations in theelectrophoretic mobility of the DNA fragments in gels, with or withoutdenaturing agents, or by direct DNA sequencing. See, e.g., Myers et al.,Science, 230: 1242 (1985), which is incorporated by reference herein inits entirety. Sequence changes at specific locations also may berevealed by nuclease protection assays, such as RNase and S1 protectionor a chemical cleavage method. See, e.g., Cotton et al., Proc. Natl.Acad. Sci., USA, 85: 4397-4401 (1985) which is incorporated by referenceherein in its entirety.

Cells carrying mutations or polymorphisms in the gene of the inventionmay also be detected at the DNA level by a variety of techniques, toallow for serotyping, for example. For example, RT-PCR can be used todetect mutations. It is particularly preferred to used RT-PCR inconjunction with automated detection systems, such as, for example,GeneScan. RNA or cDNA may also be used for the same purpose, PCR orRT-PCR. As an example, PCR primers complementary to a nucleic acidencoding histidine kinase can be used to identify and analyze.mutations. Examples of representative primers are shown below in Table1.

TABLE 1 Primers for amplification of histidine kinase polynucleotidesSEQ ID NO PRIMER SEQUENCE 3 5′-ATGAAATTTTTAAAAGATACTTCAATTGC-3′ 45′-TGCTATTCCTCCTGTTGAGATTTCAATGA-3′

These primers may also be used for amplifying histidine kinase DNAisolated from a sample derived from an individual. The invention furtherprovides these primers with 1, 2, 3 or 4 nucleotides removed from the 5′and/or the 3′ end. The primers may be used to amplify the gene isolatedfrom an infected individual such that the gene may then be subject tovarious techniques for elucidation of the DNA sequence. In this way,mutations in the DNA sequence may be detected and used to diagnoseinfection and to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStaphylococcus aureus, and most preferably disease, such as, infectionsof the upper respiratory tract (e.g., otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g.,empyema, lung abscess), cardiac (e.g., infective endocarditis),gastrointestinal (e.g., secretory diarrhoea, splenic absces,retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g.,blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal andorbital cellulitis, darcryocystitis), kidney and urinary tract (e.g.,epididymitis, intrarenal and perinephric absces, toxic shock syndrome),skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis,wound infection, bacterial myositis) bone and joint (e.g., septicarthritis, osteomyelitis), comprising determining from a sample derivedfrom an individual a increased level of expression of polynucleotidehaving the sequence of FIG. 1 [SEQ ID NO: 1]. Increased or decreasedexpression of histidine kinase polynucleotide can be measured using anyon of the methods well known in the art for the quantation ofpolynucleotides, such as, for example, amplification, PCR, RT-PCR, RNaseprotection, Northern blotting and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of histidine kinase protein compared to normalcontrol tissue samples may be used to detect the presence of aninfection, for example. Assay techniques that can be used to determinelevels of a histidine kinase protein, in a sample derived from a hostare well-known to those of skill in the art. Such assay methods includeradioimmunoassays, competitive-binding assays, Western Blot analysis andELISA assays.

Antibodies

The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. “Antibodies” as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

Antibodies generated against the polypeptides of the invention can beobtained by administering the polypeptides or epitope-bearing fragments,analogues or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique knownin the art that provides antibodies produced by continuous cell linecultures can be used. Examples include various techniques, such as thosein Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor etal., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985) which isincorporated by reference herein in its entirety.

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-histidine kinase or from naive libraries(McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J. et al.,(1992) Biotechnology 10, 779-783). The affinity of these antibodies canalso be improved by chain shuffling (Clackson, T. et al., (1991) Nature352, 624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope—termed ‘bispecific’ antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides to purify the polypeptides byaffinity chromatography.

Thus, among others, antibodies against histidine kinase-polypeptide maybe employed to treat infections, particularly bacterial infections andespecially disease, such as, infections of the upper respiratory tract(e.g., otitis media, bacterial tracheitis, acute epiglottitis,thyroiditis), lower respiratory (e.g., empyema, lung abscess), cardiac(e.g., infective endocarditis), gastrointestinal (e.g., secretorydiarrhoea, splenic absces, retroperitoneal abscess), CNS (e.g., cerebralabscess), eye (e.g., blepharitis, conjunctivitis, keratitis,endophthalmitis, preseptal and orbital cellulitis, darcryocystitis),kidney and urinary tract (e.g., epididymitis, intrarenal and perinephricabsces, toxic shock syndrome), skin (e.g., impetigo, folliculitis,cutaneous abscesses, cellulitis, wound infection, bacterial myositis)bone and joint (e.g., septic arthritis, osteomyelitis).

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term “antigenically equivalent derivative” as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm “immunologically equivalent derivative” as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be “humanized”; where thecomplimentarily determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal.,(1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem1989:264,16985), coprecipitation of DNA with calcium phosphate(Benvenisty & Reshef, PNAS, 1986:83,9551), encapsulation of DNA invarious forms of liposomes (Kaneda et al., Science 1989:243,375),particle bombardment (Tang et al., Nature 1992, 356:152, Eisenbraun etal., DNA Cell Biol 1993, 12:791) and in vivo infection using clonedretroviral vectors (Seeger et al., PNAS 1984:81,5849).

Antagonists and agonists—assays and molecules

Polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g. Coligan et al., CurrentProtocols in Immunology 1(2): Chapter 5 (1991).

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action ofhistidine kinase polypeptides or polynucleotides, particularly thosecompounds that are bacteriostatic and/or bacteriocidal, and theseeffects may be measured in assays comprising the response regulatorpolynucleotide of SEQ ID NO:5 or the polypeptide of SEQ ID NO:6. Themethod of screening may involve high-throughput techniques. For example,to screen for agonists or antagoists, a synthetic reaction mix, acellular compartment, such as a membrane, cell envelope or cell wall, ora preparation of any thereof, comprising histidine kinase polypeptideand a labeled substrate or ligand of such polypeptide is incubated inthe absence or the presence of a candidate molecule that may be ahistidine kinase agonist or antagonist. The ability of the candidatemolecule to agonize or antagonize the histidine kinase polypeptide isreflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of histidine kinasepolypeptide are most likely to be good antagonists. The method ofscreening may also comprise the response regulator polynucleotide of SEQID NO:5 or the polypeptide of SEQ ID NO:6. Molecules that bind well andincrease the rate of product production from substrate are agonists.Detection of the rate or level of production of product from substratemay be enhanced by using a reporter system. Reporter systems that may beuseful in this regard include but are not limited to colorimetriclabeled substrate converted into product, a reporter gene that isresponsive to changes in histidine kinase polynucleotide or polypeptideactivity, and binding assays known in the art.

Another example of an assay for histidine kinase antagonists is acompetitive assay that combines histidine kinase and a potentialantagonist with histidine kinase-binding molecules, recombinanthistidine kinase binding molecules, natural substrates or ligands, orsubstrate or ligand mimetics, or the response regulator polynucleotideof SEQ ID NO:5 or the polypeptide of SEQ ID NO:6, under appropriateconditions for a competitive inhibition assay. histidine kinase can belabeled, such as by radioactivity or a colorimetric compound, such thatthe number of histidine kinase molecules bound to a binding molecule orconverted to product can be determined accurately to assess theeffectiveness of the potential antagonist.

This invention provides a method of screening drugs to identify thosewhich interfere with i) the interaction of the histidine kinase with aresponse regulator, such as the response regulator of SEQ ID NO: 6, themethod comprising incubating the histidine kinase with responseregulator in the presence of the drug and measuring the ability of thedrug to block this interaction; and/or ii) the ability of the histidinekinase to autophosphorylate, the method comprising incubating thehistidine kinase with the drug and measuring the ability of the drug toprevent autophosphorylation.

The response regulator is preferably the cognate response regulator ofthe histidine kinase, or another response regulator which is capable ofusing the histidine kinase as a substrate, and is preferably fromStaphylococcus aureus but may be from another microorganism e.g.Bacillus.

The invention also relates to inhibitors identified thereby.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention, including, for example, the response regulatorpolynucleotide of SEQ ID NO:5 or the polypeptide of SEQ ID NO:6, andthereby inhibit or extinguish its activity. Potential antagonists alsomay be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a binding molecule, without inducing histidinekinase-induced activities, thereby preventing the action of histidinekinase by excluding histidine kinase from binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, JNeurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of histidine kinase.

Each of the DNA sequences provided herein may be used in the discoveryand development of antibacterial compounds. The encoded protein, uponexpression, can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgarno or other translationfacilitating sequences of the respective mRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The invention also provides the use of the polypeptide, polynucleotideor inhibitor of the invention to interfere with the initial physicalinteraction between a pathogen and mammalian host responsible forsequelae of infection. In particular the molecules of the invention maybe used: in the prevention of adhesion of bacteria, in particular grampositive bacteria, to mammalian extracellular matrix proteins onin-dwelling devices or to extracellular matrix proteins in wounds; toblock histidine kinase protein-mediated mammalian cell invasion by, forexample, initiating phosphorylation of mammalian tyrosine kinases(Rosenshine et al., Infect. Immun. 60:2211 (1992); to block bacterialadhesion between mammalian extracellular matrix proteins and bacterialhistidine kinase proteins that mediate tissue damage and; to block thenormal progression of pathogenesis in infections initiated other than bythe implantation of in-dwelling devices or by other surgical techniques.

The antagonists and agonists of the invention may be employed, forinstance, to inhibit and treat disease, such as, infections of the upperrespiratory tract (e.g., otitis media, bacterial tracheitis, acuteepiglottitis, thyroiditis), lower respiratory (e.g., empyema, lungabscess), cardiac (e.g., infective endocarditis), gastrointestinal(e.g., secretory diarrhoea, splenic absces, retroperitoneal abscess),CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellulitis,darcryocystitis), kidney and urinary tract (e.g., epididymitis,intrarenal and perinephric absces, toxic shock syndrome), skin (e.g.,impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g., septic arthritis,osteomyelitis).

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with histidine kinase, or afragment or variant thereof, adequate to produce antibody and/or T cellimmune response to protect said individual from infection, particularlybacterial infection and most particularly Staphylococcus aureusinfection. Also provided are methods whereby such immunological responseslows bacterial replication. Yet another aspect of the invention relatesto a method of inducing immunological response in an individual whichcomprises delivering to such individual a nucleic acid vector to directexpression of histidine kinase, or a fragment or a variant thereof, forexpressing histidine kinase, or a fragment or a variant thereof in vivoin order to induce an immunological response, such as, to produceantibody and/or T cell immune response, including, for example,cytokine-producing T cells or cytotoxic T cells, to protect saidindividual from disease, whether that disease is already establishedwithin the individual or not. One way of administering the gene is byaccelerating it into the desired cells as a coating on particles orotherwise.

Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid,or a DNA/RNA hybrid.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within it an immunological response, induces an immunologicalresponse in such individual to a histidine kinase or protein codedtherefrom, wherein the composition comprises a recombinant histidinekinase or protein coded therefrom comprising DNA which codes for andexpresses an antigen of said histidine kinase or protein codedtherefrom. The immunological response may be used therapeutically orprophylactically and may take the form of antibody immunity or cellularimmunity such as that arising from CTL or CD4+ T cells.

A histidine kinase polypeptide or a fragment thereof may be fused withco-protein which may not by itself produce antibodies, but is capable ofstabilizing the first protein and producing a fused protein which willhave immunogenic and protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Hemophilus influenzae, Glutathione-S-transferase(GST) or beta-galactosidase, relatively large co-proteins whichsolubilise the protein and facilitate production and purificationthereof. Moreover, the co-protein may act as an adjuvant in the sense ofproviding a generalized stimulation of the immune system. The co-proteinmay be attached to either the amino or carboxy terminus of the firstprotein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Staphylococcus aureus will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStaphylococcus aureus infection, in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation instonic with the bodily fluid, preferably the blood, of theindividual; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certainhistidine kinase protein, it is to be understood that this coversfragments of the naturally occurring protein and similar proteins withadditions, deletions or substitutions which do not substantially affectthe immunogenic properties of the recombinant protein.

Compositions, kits and administration

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or their agonists or antagonists.The polypeptides of the invention may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a polypeptide ofthe invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStaphylococcus aureus wound infections.

Many orthopaedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 μg/kg of antigen, and such dose ispreferably administered 1-3 times and with an interval of 1-3 weeks.With the indicated dose range, no adverse toxicological effects will beobserved with the compounds of the invention which would preclude theiradministration to suitable individuals.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1

Strain Selection, Library Production and Sequencing

The polynucleotide having the DNA sequence given in SEQ ID NO:1 wasobtained from a library of clones of chromosomal DNA of Staphylococcusaureus in E. coli. The sequencing data from two or more clonescontaining overlapping Staphylococcus aureus DNAs was used to constructthe contiguous DNA sequence in SEQ ID NO:1. Libraries may be prepared byroutine methods, for example: Methods 1 and 2 below.

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E. coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagedlibrary. The library is amplified by standard procedures.

6 1150 base pairs nucleic acid double linear 1 ATATTGTATT AGTCATTTTAAAAGGGCGGA ATAAAATATG AAATTTTTAA AAGATACTTC 60 AATTGCTGAA ATATCGTCTATACTTTATCT GATTTTTCCT ATTGCCGGTA TATTTTTTAA 120 TGAAGTATAT GGTCCCAAATGGTTGTATAT TATATCAGTC ATTGTCTTTT CGTTGTCGTA 180 TCTTATATTA GTTATAGTAAATAATAGACT TAATACATTA ATGTTTTACA TTTTGTTGAT 240 TATTCATTAT TTTATTATTTGTTATTTTGT TTTCAGTGTA CATCCAATGC TAAGTTTGTT 300 TTTCTTTTAT AGTGCTTTTGCCGTTCCATT TACTTTTAAA AATAATGTTA AAAAAACGGC 360 AACTAATCTT TTCATACTAACAATGATTAT ATGTACAATA ATAACGTACT TATTGTATAA 420 CAACTATTTT GTTGCAATGATGGTTTATTA TGTCGTTATA TCGTTAATAA TGCTAGATAA 480 TTTTAAAAAA ATGAAAAACCGTGAATATCA AAAAGAAATA GCAGAAAAAA ATAGACATAT 540 TAATACATTA ATTGCTGAACAAGAGCGACA TAGAATTGGT CAAGACTTAC ATGATACGTT 600 AGGGCATGTG TTTGCAAGTTTATCATTAAA ATCAGAATTA GCTTATAAAC TAATAGATAC 660 TGATGTAGAA AAAGTAAAAGCTGAATTATT AGCAATTAAT AAATTATCTC GTGAATCATT 720 GAACAAAGTT CGAGAAATTATTGATGATGT AAAATTACCA TCATTTATTG AAGAGATTGA 780 TAGTATACGT AAAGTTTTAAAAGATGCTGA TATTGATTTT ACATTTGAAA ATAAAGAATT 840 AGCGCAAGTA TTAAGTCCTACTAAACAATC TATGGTAGTT ATGATTACGC GTGAAGCGAT 900 AAATAATGTT ATTAAACATGCAAATGCTTC AAAAGTTCAT GGTAAATTAA AAACTGTAAA 960 CAATCATAAA TTACTGCTTATGATTGAAGA TGATGGCAAA GGTATCGATA GTGATTGTGA 1020 GGTGAAAAGT ATTTCACAGCGTGTACAACA TTTAAATGGA ACTTTAGCAG TCGACTCAAC 1080 AAATGGAACT AAAATAATCATTGAAATCTC AACAGGAGGA ATAGCATGAC ATCTTTAATT 1140 ATTGCAGAAG 1150 363amino acids amino acid single linear 2 Met Lys Phe Leu Lys Asp Thr SerIle Ala Glu Ile Ser Ser Ile Leu 1 5 10 15 Tyr Leu Ile Phe Pro Ile AlaGly Ile Phe Phe Asn Glu Val Tyr Gly 20 25 30 Pro Lys Trp Leu Tyr Ile IleSer Val Ile Val Phe Ser Leu Ser Tyr 35 40 45 Leu Ile Leu Val Ile Val AsnAsn Arg Leu Asn Thr Leu Met Phe Tyr 50 55 60 Ile Leu Leu Ile Ile His TyrPhe Ile Ile Cys Tyr Phe Val Phe Ser 65 70 75 80 Val His Pro Met Leu SerLeu Phe Phe Phe Tyr Ser Ala Phe Ala Val 85 90 95 Pro Phe Thr Phe Lys AsnAsn Val Lys Lys Thr Ala Thr Asn Leu Phe 100 105 110 Ile Leu Thr Met IleIle Cys Thr Ile Ile Thr Tyr Leu Leu Tyr Asn 115 120 125 Asn Tyr Phe ValAla Met Met Val Tyr Tyr Val Val Ile Ser Leu Ile 130 135 140 Met Leu AspAsn Phe Lys Lys Met Lys Asn Arg Glu Tyr Gln Lys Glu 145 150 155 160 IleAla Glu Lys Asn Arg His Ile Asn Thr Leu Ile Ala Glu Gln Glu 165 170 175Arg His Arg Ile Gly Gln Asp Leu His Asp Thr Leu Gly His Val Phe 180 185190 Ala Ser Leu Ser Leu Lys Ser Glu Leu Ala Tyr Lys Leu Ile Asp Thr 195200 205 Asp Val Glu Lys Val Lys Ala Glu Leu Leu Ala Ile Asn Lys Leu Ser210 215 220 Arg Glu Ser Leu Asn Lys Val Arg Glu Ile Ile Asp Asp Val LysLeu 225 230 235 240 Pro Ser Phe Ile Glu Glu Ile Asp Ser Ile Arg Lys ValLeu Lys Asp 245 250 255 Ala Asp Ile Asp Phe Thr Phe Glu Asn Lys Glu LeuAla Gln Val Leu 260 265 270 Ser Pro Thr Lys Gln Ser Met Val Val Met IleThr Arg Glu Ala Ile 275 280 285 Asn Asn Val Ile Lys His Ala Asn Ala SerLys Val His Gly Lys Leu 290 295 300 Lys Thr Val Asn Asn His Lys Leu LeuLeu Met Ile Glu Asp Asp Gly 305 310 315 320 Lys Gly Ile Asp Ser Asp CysGlu Val Lys Ser Ile Ser Gln Arg Val 325 330 335 Gln His Leu Asn Gly ThrLeu Ala Val Asp Ser Thr Asn Gly Thr Lys 340 345 350 Ile Ile Ile Glu IleSer Thr Gly Gly Ile Ala 355 360 29 base pairs nucleic acid single linear3 ATGAAATTTT TAAAAGATAC TTCAATTGC 29 29 base pairs nucleic acid singlelinear 4 TGCTATTCCT CCTGTTGAGA TTTCAATGA 29 850 base pairs nucleic aciddouble linear 5 TTCCCTTTTT TGTTTGTATT TAGATCCAGC CTTTTTCATT TGCCTTTTTCCAAGCATCAA 60 AACGATTATC TGCAAATAAT TTATCAATTA TAACAGATGT ATAATTTCTAACTGTTCCAT 120 CTGTCAAAAA TAATTTTTCA CTTATTTCTT TACTACTTAA ACCATTGCCAATTTCCCTTA 180 ATACAATTTG TTCTTTGGGC GTTAATGGGT TTTTATCTAC AAAAAATGAAGTCATCAATG 240 TGGCGCTATA TTCTCTCTCT CCGTTATTTA CTTTATTAAT GGTTTCCACCAATTCTTCTA 300 TCGAACGTTC TTTTAAAACA TATGCATCCA CATCATTCAC AACTGCTTTTTCAAAGTATC 360 CCGGTCTTTT AAAAGTTGTT ACAATAATCA CTTTAATATT CAAATGCTTTTTTCTAATTT 420 CCGCTAAAAC TTCAAGTCCA GTCATGCCTG GCATTTCTAT ATCTAAAATAACAACGTTAG 480 GATTATATTC TTCAATAAGT TTCATTGCAT CGAGACCATT ATCAGTATCTGCTAAAATTT 540 CAAAATCACC ATGTAGTTTA ATTAATTGAA CCATTGCCTG TCGTAACATATTTTGATCTT 600 CTGCAATAAT TAAAGATGTC ATGCTATTCC TCCTGTTGAG ATTTCAATGATTATTTTAGT 660 TCCATTTGTT GAGTCGACTG CTAAAGTTCC ATTTAAATGT TGTACACGCTGTGAAATACT 720 TTTCACCTCA CAATCACTAT CGATACCTTT GCCATCATCT TCAATCATAAGCAGTAATTT 780 ATGATTGTTT ACAGTTTTTA ATTTACCATG AACTTTTGAA GCATTTGCATGTTTAATAAC 840 ATTATTTATC 850 200 amino acids amino acid single linear 6Met Thr Ser Leu Ile Ile Ala Glu Asp Gln Asn Met Leu Arg Gln Ala 1 5 1015 Met Val Gln Leu Ile Lys Leu His Gly Asp Phe Glu Ile Leu Ala Asp 20 2530 Thr Asp Asn Gly Leu Asp Ala Met Lys Leu Ile Glu Glu Tyr Asn Pro 35 4045 Asn Val Val Ile Leu Asp Ile Glu Met Pro Gly Met Thr Gly Leu Glu 50 5560 Val Leu Ala Glu Ile Arg Lys Lys His Leu Asn Ile Lys Val Ile Ile 65 7075 80 Val Thr Thr Phe Lys Arg Pro Gly Tyr Phe Glu Lys Ala Val Val Asn 8590 95 Asp Val Asp Ala Tyr Val Leu Lys Glu Arg Ser Ile Glu Glu Leu Val100 105 110 Glu Thr Ile Asn Lys Val Asn Asn Gly Glu Arg Glu Tyr Ser AlaThr 115 120 125 Leu Met Thr Ser Phe Phe Val Asp Lys Asn Pro Leu Thr ProLys Glu 130 135 140 Gln Ile Val Leu Arg Glu Ile Gly Asn Gly Leu Ser SerLys Glu Ile 145 150 155 160 Ser Glu Lys Leu Phe Leu Thr Asp Gly Thr ValArg Asn Tyr Thr Ser 165 170 175 Val Ile Ile Asp Lys Leu Phe Ala Asp AsnArg Phe Asp Ala Trp Lys 180 185 190 Lys Ala Asn Glu Lys Gly Trp Ile 195200

What is claimed is:
 1. An isolated polypeptide comprising SEQ ID NO:2.2. A composition comprising the isolated polypeptide of claim 1 and acarrier.
 3. The isolated polypeptide of claim 1, wherein the isolatedpolypeptide consists of SEQ ID NO:2.
 4. A composition comprising theisolated fusion polypeptide of claim 1 and a carrier.
 5. An isolatedfusion polypeptide comprising a heterologous amino acid sequence fusedto SEQ ID NO:2.
 6. A composition comprising the isolated fusionpolypeptide of claim 5 and a carrier.
 7. A composition comprising theisolated polypeptide of claim 5 and a carrier.
 8. A compositioncomprising the isolated polypeptide of claim 5 and a carrier.
 9. Anisolated polypeptide comprising at least 50 consecutive amino acids ofSEQ ID NO: 2, wherein said polypeptide is capable of functioning as ahistidine kinase.
 10. An isolated fusion polypeptide comprising aheterologous amino acid sequence fused to at least 50 consecutive aminoacids of SEQ ID NO: 2, wherein said polypeptide is capable offunctioning as a histidine kinase.
 11. An isolated polypeptidecomprising at least 30 consecutive amino acids of SEQ ID NO: 2, whereinsaid polypeptide is capable of functioning as a histidine kinase.
 12. Acomposition comprising the isolated polypeptide of claim 11 and acarrier.
 13. An isolated fusion polypeptide comprising a heterologousamino acid sequence fused to at least 50 consecutive amino acids of SEQID NO: 2, wherein said polypeptide is capable of functioning as ahistidine kinase.
 14. A composition comprising the isolated fusionpolypeptide of claim 13 and a carrier.